6-{8 n-(substituted-imidoyl)-aminoacetamido{9 pencillanic acids

ABSTRACT

6-(N-(substituted-imidoyl)aminoacetamido)penicillanic acids and their salts are valuable as antibacterial agents, nutritional supplements in animal feeds, therapeutic agents in poultry and animals, including man, and are especially useful in the treatment of infectious diseases caused by Gram-positive and certain Gram-negative bacteria, most particularly those caused by the Proteus genus. 6-(N-(Phenylacetimidoyl)aminoacetamido)penicillanic acid, a preferred embodiment of the invention, is prepared by Raney nickel hydrogenation (50 p.s.i., R.T.) of an aqueous solution of sodium 6-(3-benzyl-1,2,4oxadiazole-5-one-4-acetamido)penicillanate which is prepared in turn by reaction of 6-aminopenicillanic acid with 3-benzyl-1,2,4oxadiazole-5-one-4-acetyl chloride.

United States Patent Holdrege 1 June 13, 19726-[N-(SUBSTITUTED-IMIDOYL)- AMINOACETAMIDO]PENCILLANIC ACIDS [72]Inventor: Charles Truman Holdrege, Camillus, NY.

[73] Assignee: Bristol-Myers Company, New York, N .Y.

[22] Filed: June 29, 1970 [21] Appl. No.: 50,997

[5 2] US. Cl ..260/239.l, 424/271 [51] ..C07d 99/14, CO7d 99/24 [58]Field of Search ..260/239.1

[56] References Cited UNITED STATES PATENTS 3,454,557 7/1969 Patchett etal ..260/239.1 3,453,265 7/1969 Patchett et a1 ..260/239.1 3,479,3391/1969 Holdrege ...260/239.1 3,481,922 12/1969 Holdrege ..260/239.l

Richard H. Brink and James Magee, Jr.

[57] ABSTRACT 6-[N-(substituted-imidoyl)aminoacetamido]penicillanicacids and their salts are valuable as antibacterial agents, nutritionalsupplements in animal feeds, therapeutic agents in poultry and animals,including man, and are especially useful in the treatment of infectiousdiseases caused by Gram-positive and certain Gram-negative bacteria,most particularly those caused by the Proteus genus.G-[N-(Phenylacetimidoyl)- aminoacetamido]penicillanic acid, a preferredembodiment of the invention, is prepared by Raney nickel hydrogenation(5O p.s.i., RT.) of an aqueous solution of sodium6-(.3-benzyll,2,4-oxadiazole-5-one-4-acetamido)penicillanate which isprepared in turn by reaction of -aminopenicillanic acid with 3-benzyll,2,4-oxadiazole-5-one-4-acetyl chloride.

12 Claims, N0 Drawings 6-[N-(SUBSTITUTED-IM1DOYIJ-AMINOACETAMIDOPENCILLANIC ACIDS BACKGROUND OF THE INVENTION 1. Field ofthe Invention The penicillins of the present invention possess the usualattributes of such compounds and are particularly useful in thetreatment of infections caused by Proteus and certain otherGram-negative bacteria.

2. Description of the Prior Art Heretofore very few penicillins havebeen found to possess any significant activity against Proteus. Thecompounds of the present invention exhibit this desirable activity atsignificantly low concentrations.

Aminomethylpenicillin and its N-carbobenzyloxy derivative are known fromU. S. Pat. Nos. 3,120,514 and 2,941,995 respectively; see also U. S.Pat. No. 3,453,263. For aguanidinomethylpenicillin see U. S. Pat. Nos.3,406,185 and 3,454,557.

a-Aminobenzylpenicillins and a-aminothenylpenicilliris are known, asfrom U. S. Pat. Nos. 2,985,648, 3,140,282, 3,373,156, 3,308,023 and3,342,677 and British Pat. Nos. 903,785, 918,169, 978,178, 991,586 and1,033,257 and for such compounds bearing substituents on the a-aminogroup from U. S. Pat. Nos. 3,198,804, 3,308,023, 3,381,001 3,448,104,3,453,264, 3,471,474, 3,479,339, 3,481,922 and 3,483,188 and from suchBritish Pat. as Nos. 891,777, 894,457, 1,040,166, 1,048,907, 1,051,675,1,053,415, 1,053,818, 1,057,697, 1,064,893, 1,066,107, 1,080,247,1,125,339, 1,130,445, 1,134,237 and 1,138,745 and from South Africa Pat.No. 67/2092. N-Substituted 6-ureidopenicillanic acids have beendisclosed, for example in U. S. Pat. Nos. 3,118,877, 3,120,512 and3,180,863 and in Belgian Patent 603,703 and German Pat. No. 1,120,072;6-ureidopenicil1anic acid itself is disclosed in German Pat. Nos.1,141,640 and 1,120,072 and French Pat. No. 1,324,918. A few substituteda-ureidomethylpenicillins are disclosed in U. S. Pat. No. 3,352,851 andBritish Pat. No. 1,040,166. British Pat. No. 1 ,061,335 discloses 6-(D-a-hydrazinocarbonylamino-a-phenyl-acetamido)penicillanic acid and6-(D-a-benzyloxycarbonylhydrazinocarbonylamino-a-phenylacetamido)penicillanicacid and, in general. other so-called acyl groups in place of thebenzyloxycarbonyl group.

A variety of a-guanidino-arylmethylpenicillins includingaguanidinomethylpenicillin are disclosed in U.S. Pat. Nos. 3,406,185,3,454,557 and 3,479,401 and British Pat. No. 1,164,457.

With regard to the penicillins used as intermediates in the presentinvention, numerous methylpenicillins having attached at the alphaposition a heterocyclic ring containing two or three or fourhetero-atoms and often other substituents on the heterocyclic nucleusare reported in the literature, e.g. in U. S. Pats. classified insub-class 260-2391 as exemplified most recently by U. S. Pat. Nos.3,476,743, 3,468,874, 3,427,302, 3,322,751, 3,322,750 and 3,296,250.

N-Benzoylaminomethylpenicillin was reported in South African Pat. No.60/2882 to Lovens and N-phenylacetylaminomethylpenicillin was disclosedin Zhur. Org. Khim 1(2), 348-352 (1965).

SUMMARY OF THE INVENTION S OH; ZCNHCHzi 3NH-CHC \CZCH3 NHO=C-III(IJHCOQH (I) and o s CH; 7. N -(-1l ii-Niwfill 6 F CH l]'NCIICOQII 0 0 11 wherein Z is (lower)alkyl, cycloalkyl having 4, 5, 6or 7 carbon atoms, monohalo(1ower)a1kyl, dichloromethyl,trichloromethyl, (lower)alkenyl of two to six carbon atoms,

wherein n is zero or one and R, R and R each is hydrogen, nitro,di(lower)alky1amino, (lower)alkanoylamino, (lower)- alkanoyloxy,(lower)alkyl (comprising straight and branched chain saturated aliphaticgroups having from one to six carbon atoms inclusive), amino, hydroxy,(lower)alkylthio, (lower)- alkoxy, sulfamyl, chloro, iodo, bromo, fluoroor trifluoromethyl; and nontoxic, pharmaceutically acceptable saltsthereof, e. g., with a metallic cation such as sodium, potassium,calcium, aluminum, and the like, the ammonium cation and substitutedammonium cations, e.g., cations of such nontoxic amines astri(lower)alkylamines, i.e., triethylamine, etc., procaine,dibenzylamine, N-benzyl-beta-phenethylamine, 1- ephenamine,N,N'-dibenzylethylenediamine, dehydroabietylamine,N,N-bisdehydroabietylethylenediamine, N-(1ower)alkylpiperidine, e.g.,N-ethylpiperidine, or other such amines which have been used to formpharmaceutically acceptable nontoxic salts with benzylpenicillin. Suchsalts are preferably made by treatment of the free acid (zwitterion inthe compounds of formula 1) form of the product with a strong base. Inaddition the compounds of formula 1 form acid addition salts includingthe mineral acid addition salts such as the hydrochloride, hydrobromide,hydroiodide, sulfate, sulfamate and phosphate and the organic acidaddition salts such as the maleate, fumarate, malate, mandelate,ascorbate, para-toluenesulfonate, B-naphthalenesulfonate, and the like.

In the preferred embodiments of this invention the compounds exist inthe zwitterion form and Z is phenyl, benzyl, thenyl, thienyl, furyl or(lower)alkyl and preferably, benzyl, 2- thenyl or 3-thenyl.

DETAILED DESCRIPTION Antibacterial agents such as ampicillin (U. S. Pat.No. 2,985,648) have proved highly effective in the past in the therapyof infections due to Gram-positive and Gram-negative bacteria but thesecompounds have been notably lacking in their ability to effectivelycontrol Proteus infections.

It was an object of the present invention to provide compounds useful inthe treatment of infections caused by Grampositive and Gram-negativebacteria, including particularly those caused by Proteus.

The compounds of the present invention as defined above under Summary ofthe Invention" are particularly useful in that they possessantibacterial activity against both Grampositive and Gramnegativebacteria, and most particularly exhibit activity against Proteusinfections.

Included within the scope of the present invention are easily hydrolyzedesters which are converted to the free acid form by chemical orenzymatic hydrolysis, as illustrated in Belgian Pat. No. 684,288 and inU. S. Pat. No. 3,399,207.

The penicillins of the present invention containing the a-( 3-substituted-l ,2,4-oxadiazol-5-one-4-yl) group are primarily useful asintermediates for the preparation of the corresponding penicillinscontaining the 6- N-(substitutedimidoyl)aminomethyl side chain. Thelatter are far more potent antibacterial agents.

In the treatment of bacterial infections in man, the penicillins of thisinvention containing the latter side chain are administered topically,orally and parenterally in accordance with conventional procedures forantibiotic administration in an amount of from about 5 to 125 mg./kgldayand preferably in therange of to 50 mg./kg./day for Proteus infectionsin divided dosages, e.g., three or four times a day. They areadministered in dosage units containing, for example, 125, 250, 500,1,000 and 2,000 mg. of active ingredient with suitable physiologicallyacceptable carriers or excipients. The dosage units can be in the formof liquid preparations such as solutions, dispersions, emulsions or insolid form such as tablets, capsules, etc.

The compounds of the present invention are also useful fordecontamination of filling machines and containers used for cosmeticsand topical drugs to prevent or at least reduce the very commoncontamination of such products with Proteus. In such instance, use ismade of an aqueous solution having a concentration of at least 1mgm./ml. and a contact time of at least one hour.

The compounds of Formula II above are prepared by the process whichcomprises coupling with 6-aminopenicillanic acid in the usual manner anacid of the formula --O Wi e preferably as the acid chloride, wherein 2has the meaning set out above.

The compounds of Formula II above are converted into those of Formula Iby hydrogenation and preferably by hydrogenation at about roomtemperature over Raney nickel catalyst of an aqueous solution of a saltof the compound of Formula II and preferably a sodium or potassium salt.The hydrogen pressure is preferably about 50 p.s.i. or higher.

The starting acids of formula Ill are prepared by standard methods ofsynthesis from known nitriles by the following reaction scheme wherein 2has the meaning set out above (and as illustrated in the Examplesbelow):

per Beil. 9 304, II 214 and references therein and in Chem. Rev. 62, 155(1962) Additional examples of such intermediates found in the literatureinclude the following:

Beil. 27, 644, II 698 and references }UN therein.

C'.A. 62,15283 b; N h. Appl. 6,402,833;

C.A. 68, P952329 t.

Beil. 27, II no.

Ber. 22, 2399.

CHaO

04A. 68, 21886 n; C.A. 69, 96597 (LA. 68, 21886 n; C.A. 69, 0659? y.

Structure H References Do. --N ,1 1

(LA. 68, 21886 11; C.A. (19, 96597 y;

Berichtc, 22, 2418 (1889).

(1954); Campaigne, 70, 155 (1948).

Blickc, Leonard, JACS, 68, 1034 (1046); Clrowc, Nord, J. Org. Chem., 1

Commercially available (Aldrich).

Ber. 14, 1058 (1881).

Prijs Helv. Chim. Acta., 31, 511 i948).

/\ Commercially available (Aldrich); --C11 CN Burger, Walter, JACS, 72,1988 (1950).

Winterield, Flick, Arch. Pharm. 26, 448 (1956); Sperber et al., JACS,73, 5752 (1951).

Commercially available (Aldrich).

Campaiguc, McCarthytgigg,

Aromatic and aliphatic nitriles are well-known compounds which areprepared, for example, by the methods outlined in classic textbooks suchas Organic Preparations by Conrad Weygand, lnterscience Publishers,Inc., New York (1945) on pages 115, 255 and 367 and the Chemistry of theCarbon Compounds by Victor Van Richter edited by Richard Anschutz, ThirdEnglish Edition, Elsevier Publishing Co., Inc., New York, N.Y. (1946) inVolume 111 at pages 304-306 and in Volume 1 at pages 325-327 andLaboratory Methods of Organic Chemistry by L. Gattermann revised by H.Wieland, translated from the 24th edition, MacMillan and Co., Limited.London (1943) at pages 137-138 and by the following procedure:

Z-Furarnide (11.1 g., 0.1 mole, Eastman Kodak Co.) and phosphoruspentoxide (21 g., 0.15 mole) were mixed and heated with a Meeker burner(gently at first and then more strongly) until 2-cyanofuran distill at130 140 C., 5.6 g.

Such amides are prepared by published methods as illustrated in thefollowing table in which it is to be understood that acids are convertedto acid chlorides by treatment with thionyl chloride and acid chloridesare converted to amides by reaction with ammonia;

Amlde Illustrative Sources C ONHz Acid, acid chloride, amide: Looker,Wilson, J. Heterocychc Chem. 2 (4). 348 (1965). Acld, S acid chloride,ethyl ester: Hurd, N Mori, J. Am. Chem. Soc. 11,

--C ONHz Acid, ethyl ester, amide". U.S. 1 1L 3,060,187.

Acid, ethyl ester: Bell. 27, 317.

Amide' C.A. 58' 3409 Kochetkov, Sok olov; Luboshnlkova, Zh. Obshch.Khlm. 32, 1778- (1962).

Quilico, Panizzi, Gazz. Qhim.

Ital. 72, 458 (1942). Quihco, Stagno dAlcontres, Gezz. Chim. Ital. 79,654 (1949). Mina, Rateb, Soliman, J. Chem. Soc. 1962, 4234.

Acid, acid chloride: Doyle and Nayler, U.S. 2 996,501 and referencescited therein.

Alternative Method for Preparing Compounds of General Formula IIPenicillins of general formula II can be prepared by reaction of6-bromoacetamidopenicillanic acid with a 3-substituted- 1,2,4-oxadiazole-5-one as follows:

lBaseTEA, or 2,6-lutidine Solvent-THE, dioxane, CHzCh, 011013, or DMF zN CH CONH s\ CH3 L 2 7 orn N y io n 0 N 00011 176.1 g. (1.61 mole) ofZ-thienonitrile, 11.9 g. (1.61 mole) of hydroxylarnine hydrochloride,85.3 g. (0.805 mole) of sodium carbonate, 1 liter of percent ethanol and300 m1. of water was heated at reflux for 17 hours. A small amount ofsolid was removed by filtration. Additional water was added to thefiltrate and the ethanol was stripped off at reduced pressure causingthe crystalline product to separate. The filtered product was washedwith water. The crude product was heated with benzene most of it goinginto solution. Cooling this mixture gave afier filtration and drying170.9 g. of 2- thiophenecarboxamine; m.p. 87-8 8 (lit. ref. Beil. 18,290).

Ethyl chloroformate (130.2 g., 1.2 mole) was added gradually to a heatedand stirred solution of 170.9 g. (1.2 mole) of Z-thiophenecarboxamide,96.5 ml. of pyridine and 300 ml. of benzene. An exothermic reaction tookplace which caused the mixture to reflux. The mixture was refluxed for15 minutes after the addition of ethyl chloroformate was complete. Themixture was cooled slightly and 600 ml. of water was added. The benzenewas distilled off and the remaining aqueous mixture was refluxed for 2.5hours. After cooling overnight the product was filtered and washed withwater. The crude product was dissolved in dilute aqueous sodiumhydroxide, the solution carbon treated, and reprecipitated byacidification with acetic acid. There was obtained 139.4 g. of-hydroxy-3-(2'-thienyl)-1,2,4-oxadiazole; m.p. 185-190 dec. The nuclearmagnetic resonance and infrared spectra were consistent for the desiredcompound.

3-(2'-F uryl)-5-hydroxy-1 ,2 ,4-oxadiazole. A mixture of 69.5 g., (0.746mole) of 2-furylnitrile [Ber. 14, 1058 (1881)], 51.8 g. (0.746 mole) ofhydroxylamine hydrochloride, 39.5 g. (0.373 mole) of sodium carbonate,500 ml. of 95 percent ethanol and 150 ml. of water was refluxedovernight. The mixture was filtered and 150 ml. of water was added. Theethanol was stripped off at reduced pressure. The aqueous phase wasextracted several times with ethyl acetate. The combined organicextracts were carbon treated, dried with sodium sulfate and the solventremoved at reduced pressure leaving 88.9 g. of Z-furylamide oxime as aviscous oil (lit. ref. CA. 52, 7292b).

Ethyl chloroformate (76.5 g., 0.705 mole) was added slowly to a heatedand stirred solution of 88.9 g. of Z-furylamide oxime, 56.7 ml. (0.705mole) of pyridine and 200 ml. of benzene. After the addition of ethylchloroformate was complete the reaction mixture was refluxed anadditional minutes. Water (400 ml.) was added and the benzene distilledoff. The remaining aqueous mixture was refluxed for 2.5 3 hours. Aftercooling in an ice bath the solid product was collected by filtration andwashed with water. The product was dissolved in dilute aqueous sodiumhydroxide solution, carbon treated, the solution acidified with aceticacid with ice cooling and the reprecipitated product filtered and washedcarefully with ice water. The product was again purified in the samemanner giving after drying in vacuo over phosphorus pentoxide 50.1 g. of3-(2'-furyl)-5-hydroxy-1,2,4-oxadiazole; dec. above 210.

Anal. Calcd. for C H N,O,: C, 47.37; H, 2.65; N, 18.42. Found: C, 47.27;H, 2.91; N, 18.39.

Illustrative examples of the preparation of penicillins of the presentinvention follow. These examples are given in illustration of, but notin limitation of, the present invention. All temperatures are in degreesCentigrade. Skellysolve B" is a petroleum ether fraction of b.p. 60 68C. consisting essentially of n-hexane.

DESCRIPTION OF THE PREFERRED EMBODIMENTS mide oxime was dried withsodium sulfate, filtered, and concentrated to a volume of about 400 m1.Reference to phenylacetamide oxime: Beil. 9, 446,11 305.

Ethyl chloroformate (96 ml., 1 mole) was added gradually from a droppingfunnel to the above prepared solution of phenylacetarnide oxime inbenzene plus 80.5 ml. (1 mole) of pyridine initially at roomtemperature. The exothermic reaction caused the mixture to reflux. Themixture was refluxed for 15 minutes longer, cooled somewhat and 500 ml.of water added. The benzene was distilled off and the remaining aq ueousmixture was heated at reflux for 2.5 hours longer. The oil whichseparated was extracted into benzene. The benzene phase was extractedwith 175 ml. of 20 percent sodium hydroxide solution and then with 50ml. of water. The combined basic extracts were acidified with aceticacid giving oily crystals. The crude product was recrystallized frombenzene; m.p. 94, yield 41 g. The product was combined with 100 ml. ofwater and 50 ml. of 20 percent sodium hydroxide solution and the mixtureheated for one hour on the steam bath. The cooled solution was acidifiedwith acetic acid. An additional 100 ml. of water was added to make themixture more fluid. The product was collected by filtration. The dampfilter cake was dissolved in about 500 ml. of chloroform with warming.The solution was dried with sodium sulfate, filtered, and the solventdistilled off at reduced pressure. The residue was extracted with three250 ml. portions of boiling cyclohexane and with 500 ml. of boiling 1:1cyclohexane-benzene. The insoluble residue was slurried with cyclohexaneand collected by filtration. There was obtained 25.2 g. of3-benzyl-l,2,4oxadiazole-S-one; m.p. 109"112 (Beil. 27, 11 710 reportsm.p. The infrared spectrum was consistent for the desired product.3-Benzyl- 1 ,2,4-oxadiazole-5 -one-4-acetic acid A solution of 25 g.(0.142 mole) 3-benzyl-l ,2,4-oxadiazole- 5-one in 300 ml. of methanolwas stirred with 20 g. of powdered Molecular Sieves (Linde 4A) for onehour at room temperature. The Molecular Sieves were removed byfiltration. To the stirred filtrate was added 8.45 g. (0.156 mole) ofsodium methoxide and then 26.2 g. (0.156 mole) of ethyl bromoacetate.The mixture was heated at reflux for 18 hours. The solvent was distilledoff at reduced pressure. To the residue was added 200 ml. of acetic acidand 100 ml. of 6 N hydrochloric acid and the mixture refluxed for 1.5hours. The solvents were distilled off at reduced pressure. To theresidue was added water and benzene causing the product to crystallize.The product was filtered, washed on the filter with water and benzeneand air dried. The crude product was recrystallized from toluene with acarbon treatment giving 15.9 g. of white crystalline product afterdrying in vacuo over phosphorus pentoxide; m.p. ll1-113. The infraredand nuclear magnetic resonance spectra were consistent for the desiredproduct.

Anal Calcd. for C I- 14 0 C, 56.41; H, 4.30, N, 11.96. Found: C, 56.61;H, 4.42; N, 12.21.

3-Benzyl-l ,2,4-oxadiazole-5-one-4-acetyl chloride A solution of 5.0 g.(0.0214 mole) of 3-benzyl-l,2,4-oxadiazole-5-one-4-acetic acid in 200ml. of methylene chloride was gassed briefly (2 minutes) with hydrogenchloride. To the resulting clear solution was added 5.8 g. (0.0278 mole)of phosphorus pentachloride (efifervescence) and the mixture stored atroom temperature for one hour with occasional swirling. The solvent wasstripped off at reduced pressure. The crystalline residue was trituratedwith cyclohexane and dried at reduced pressure. The infrared spectrum(Nujol) was consistent for the desired acid chloride. Sodium 6-(3-benzyl- 1 ,2,4-oxadiazole-5-one-4-acetarnido)- penicillanate Asolution of 3-benzyl-1,2,4-oxadiazole-5-one-4-acetyl chloride (obtainedfrom 5 g. of the acid) in 90 ml. of acetone was added to a rapidlystirred mixture of 4.62 g. of 6 arninopenicillanic acid, 7.2 g. ofsodium bicarbonate, ml. of water and 60 m1. of acetone initially at 5.The cooling bath was removed and the mixture was stirred for 45 minutesand extracted three times with ethyl acetate. The aqueous phase waslayered with ethyl acetate and acidified with 42% phosphoric acid. Twomore extractions on the aqueous phase were made with ethyl acetate. Thecombined ethyl acetate extracts were washed three times with water,dried with sodium sulfate, filtered and treated with 7.6 ml. (0.02 mole)of sodium Z-ethylhexanoate in l-butanol. The mixture was concentratedsomewhat and the crystalline product filtered and washed with ethylacetate (solid A); yield 4.1 g. The filtrate was stripped of solvent andthe residue triturated with anhydrous ether giving 3.2 g. of sodium6-(3-benzyl-l,2,4-oxadiazole-5-one-4- acetamido)-penicillanate;decomposes 170-180 with prior darkening above about 160. The infraredand nuclear megnetic resonance spectra were consistent for the assignedstructure.

Anal. Calcd. for C H N O SNa: C, 50.21; H, 4.21;

Found:

Solid A was extracted at room temperature with 200 ml. of acetone. Theinsoluble material, which was mostly the sodium salt of the side chainacid, was removed by filtration. The filtrate was stripped of solvent atreduced pressure and the residue triturated with anhydrous ether giving2.5 g. of sodium 6-( 3 -benzyl-l ,2 ,4-oxadiazole-5-one-4-acetamido)penicillanate. 6-[N-(Phenylacetimidoyl)aminoacetamidolpenicillanic acid.

A solution of 5.5 g. of sodium 6-(3-benzyl-1,2,4-oxadiazole-5-one-4-acetamido)penicillanate in 150 ml. of water plus 50 m1. ofdioxane was hydrogenated in the presence of commercial Raney nickelcatalyst (No. 28) in a Parr hydrogenation apparatus at an initialpressure of 50 psi. at room temperature for 2 hours, the theoreticalamount of hydrogen being taken up. The catalyst was removed byfiltration. The filtrate was adjusted to pH 4.5 with 6 N hydrochloricacid and the clear solution concentrated to a small volume. Theconcentrate was adjusted to pH 2 and then to pH 4.2 with percent sodiumhydroxide and concentrated to dryness. Ethyl acetate was added to theresidue and this stripped out. The residue was dried in vacuo overphosphorus pentoxide. The product was dissolved with 70 ml. of 95percent ethanol the insoluble material being removed by filtration. Thesolution was diluted to the cloud point with acetone causingcrystallization of the product. The product was filtered, washed withacetone, dried in vacuo over phosphorus pentoxide and further dried in avacuum oven at 65 for 3 hours; yield 2.4 g., decomposes l80185 withprior darkening above about 160. The infrared and nuclear magneticresonance spectra were fully consistent for the desired product.

Anal Calcd. for

C I-1 N 05: C, 55.37; H, 5.68; N, 14.35. Found: C, 54.82; H, 6.13;

54.80 6.20 N, 13.88, 1-1,0, 1.2. 13.89 1.4. Found values corrected for1.2 and 1.4% 11,0:

EXAMPLE 2 5-Hydroxy-3-phenyl-1 ,2,4-oxadiazo1e A solution of 103.1 g. (1mole) of benzonitrile in 400 ml. of 95 percent ethanol was added to amixture of 53 g. (0.5 mole) of sodium carbonate, 69.5 g. (1 mole) ofhydroxylarnine hydrochloride, 200 ml. of water and 300 ml. of 95 percentethanol. The mixture was refluxed for 18 hours. Most of the ethanol wasdistilled off and additional water was added. The product, whichcrystallized on cooling, was filtered, washed with water, and air dried.The product was dissolved in benzene and the benzene distilled off toremove any remaining water leaving a crystalline residue of benzamideoxime (lit. ref. Beil. 9, 304,11214).

Ethyl chloroformate (96 m1., 1 mole) was added gradually from a droppingfunnel to a stirred solution of benzamide oxirne in 250 m1. of warmbenzene plus 80.5 m1. (1 mole) of pyridine. The exothermic reactioncaused the reaction mixture to reflux. The mixture was refluxed for 15minutes longer, cooled somewhat, and 500 ml. of water added. The benzenewas distilled off. The aqueous mixture remaining was refluxed for 2.5hours. After cooling in an ice bath the crystalline product wascollected by filtration. The crude product was purified by dissolving indilute aqueous sodium hydroxide and acidifying with acetic acid; yield122.9 g., m.p. 196200 (lit ref. Beil. 27, 644,11698). 3-Phenyl-1,2,4-oxadiazo1e-5-one-4-acetic acid A mixture of 32.2 g., (0.193 mole)of ethyl bromoacetate, 10.5 g. of sodium methoxide, 28.5 g. (0.176 mole)of 5- hydroxy-3-phenyl-1,2,4-oxadiazole and 300 ml. of methanol washeated at reflux for 4 hours and then stored at room temperatureovernight. To the reaction mixture was added 10 g. of sodium hydroxideand 30 m1. of water. The solution was heated at reflux for 1.5 hours.Water (70 ml.) was added and the methanol distilled off at reducedpressure. Acidification of the aqueous concentrate with 6 N hydrochloricacid gave an oily crystalline precipitate which was filtered and airdried (solid A); yield 29 g. A second crop of product precipitated fromthe filtrate (solid B); yield 2.1 g. The infrared spectrum of solid 13was consistent for 3-phenyl-1,2,4-oxadiazole-5-one- 4-acetic acid.

Solid A was recrystallized from 1:1 percent ethanol water with a carbontreatment giving 7.8 g. of 5-hydroxy-3- phenyl-l,-2,4-oxadiazole as thefirst crop. The filtrate deposited a second crop (8.9 g.) (solid C)which had an infrared spectrum consistent for3-phenyl-1,2,4-oxadiazole-5- one-4-acetic acid. Solid C wasrecrystallized by dissolving in ethyl acetate and diluting with a largevolume of Skellysolve B." The material which immediately separated(solid D) was removed. The filtrate was stored in the cold to allow theproduct to crystallize; yield 3.8 g. The infrared and nuclear magneticresonance spectra were fully consistent for 3-phenyl- 1,2,4-oxadiazole-5-one-4-acetic acid.

Anal. Calcd. for c,,,i-t,N,o,-%H,o: c, 52.40; H, 3.96;

N, 12.23; E 0, 3.93. c, 52.37; H, 4.76; N, 12.50; H2O, 3.26. Solid D waspurified with ethyl acetate-Skellysolve B as described giving additionalproduct. The filtrate from solid C was concentrated to dryness and theresidue purified with ethyl acetate-Skellysolve B. Thus an additional2.8 g. of 3- phenyl-l ,2,4-oxadiazole-5-one-4-acetic acid was obtained.3-Phenyl-1 ,2,4-oxadiazole-5-one-4-acetyl chloride A suspension of 1.0g. (0.00455 mole) of 3-phenyl-1,2,4-oxadiazole-5-one-4-acetic acid in 50ml. of methylene chloride was gassed briefly with hydrogen chloride and1.1 g. (0.00528 mole) of phosphorus pentachloride added. The mixture wasstored at room temperature for 2 hours. The solvent was distilled fromthe resulting clear solution leaving the acid chloride as an oil. Sodium6-( 3-phenyl- 1 ,2 ,4 oxadiazole-5 -one-4- acetamido)penicillanate Asolution of 3-phenyl-l,2,4-oxadiazole-5-one-4-acety1 chloride (preparedfrom 1 g. of the acid) in 10 ml. of acetone was added to a solution of0.98 g. of 6-aminopenicillanic acid, 1.5 g. of sodium bicarbonate, 30ml. of water and 20 ml. of acetone at room temperature. The reactionmixture was stirred for 0.5 hour, diluted with 30 m1. of water andextracted once with ethyl acetate. The aqueous phase was acidified with42 percent phosphoric acid and extracted three times with ethyl acetate.The combined ethyl acetate extracts were washed three times with water,dried with sodium sulfate, filtered, concentrated somewhat and treatedwith 1.7 ml. (0.00455 mole) of sodium 2-ethyl hexanoate in l-butanol. Asmall amount of precipitate which had separated at once was removed byfiltration. A solid crystallized from the filtrate which was identifiedas being primarily the sodium salt of the side chain acid. The filtratewas stripped of solvent and the Found Anal. Calcd. for C H N O SNa: C,49.09; H, 3.89;

N, 12.72. C, 44.94;! 1, 4.69; N, 12.35; E 0, 9.77. Found valuescorrected for 9.77% E C, 48.75; H, 3.61; N, 13.40.

Found:

6-[N-( Benzimidoyl )aminoacetamido]penicillanic acid A solution of 0.74g. of sodium 6-(3-phenyl-l,2,4-oxadiazole-5-one-4-acetamido)-penicillanate in 100 ml. of water washydrogenated in the presence of commercial Raney nickel catalyst on aParr hydrogenation apparatus at room temperature at an initial pressureof 50 p.s.i. for 1.5 hours. The catalyst was removed by filtration. Thefiltrate was adjusted to pH 4.5 with 6 N hydrochloric acid, concentratedto a small volume and readjusted to pH 4.5. The solution was stripped todryness. The residue was triturated with anhydrous ether, filtered anddried in vacuo over phosphorus pentoxide; yield 0.57 g., graduallydecomposes above about 165. The infrared and nuclear magnetic resonancespectra were consistent for 6-[N-(benzimidoyl)aminoacetamido]gqpenicillanic acid.

EXAMPLE 3 3-( p-Chlorobenzyl)-l ,2,4-oxadiazo1e-5-one A solution of151.6 g. (1 mole) of p-chlorophenylacetonitrile in 200 m1. of 95 percentethanol was added to a stirred mixture of 69.5 g. (1 mole) ofhydroxylamine hydrochloride, 53 g. (0.5 mole) of sodium carbonate, 200ml. of water and 400 ml. of 95 percent ethanol. The mixture was heatedat reflux for 19 hours. Most of the ethanol was stripped ofi at reducedpressure causing the crude p-chlorophenylacetamide oxime to separate asan oil. The product was extracted into 600 ml. of benzene. The benzeneextract was washed two times with water. The product which started tocrystallize from the benzene phase was kept in solution by warming. Thebenzene solution was concentrated to about 400 ml.

Ethyl chloroformate (108.5 g., 1 mole) was added dropwise to a stirredmixture of the above prepared solution of pchlorophenylacetamide oximein benzene plus 80.5 ml. (1 mole) of pyridine. The reaction mixture wasbrought to reflux after about three-quarters of the ethyl chloroformatehad been added. The mixture was refluxed for 25 minutes after theaddition was completed. The mixture was cooled to room temperature and500 ml. of water were added. The benzene was distilled off. The aqueousresidue was heated at refiux for 2 hours. The reaction mixture wascooled in an ice bath and the aqueous phase decanted from the oil. Tothe residual oil was added a solution of 45 g. of sodium hydroxide in500 ml. of water and the mixture refluxed for 2 hours. The cooled mix-.ture was adjusted to pH 3-4 with concentrated hydrochloric acid. Theproduct was collected by filtration and washed with water. The productwas dissolved in 500 ml. of boiling benzene, the hot solution filteredand the filtrate diluted with 500 ml. of cyclohexane giving 56 g. ofproduct. The crude product was recrystallized from 1:1 95 percentethanol-water with a carbon treatment and then from benzene. The productwas dissolved in dilute aqueous sodium hydroxide and the filteredsolution was acidified with glacial acetic acid; m.p. 168-172, yield,after drying in vacuo over phosphorus pentoxide, 21.6 g. The infraredand nuclear magnetic resonance spectra were consistent for3-(p-chlorobenzyl)- 1,2,4-oxadiazole-S-one.

Anal. Calcd. for

CBH,CZN,O,: c. 5132,11, 3.35;N,

Found: C, 51.46; H, 3.63; N,

The combined benzene filtrates deposited a second crop of product whichwas purified by dissolving in dilute sodium hydroxide andreprecipitating with acetic acid; yield 3.2 g., m.p. 166-170.

3-( p-Chlorobenzyl)- l ,2,4-oxadiazole-5-one-4-acetic acid A solution of23.2 g. (0.0992 mole) of 3-(p-chlorobenzyl)- 1,2,4-oxadiazole-5-one in450 ml. of methanol was stirred at room temperature with 20 g. ofpowdered Molecular Sieves (Linde 4A) for 1.5 hours. The Molecular Sieveswere removed by filtration. The filtrate was added to a freshly preparedsolution of sodium methoxide in methanol made by adding 2.5 g. (0.1 1mole) of sodium to ml. of methanol. Ethyl bromoacetate (18.4 g., 0.11mole) was added. The stirred mixture was refluxed for 19 hours in anapparatus protected from atmospheric moisture. The solvent was distilledoff at reduced pressure. To the residue was added 200 ml. of glacialacetic acid and 100 ml. of 6 N hydrochloric acid and the mixture heatedat reflux for 2 hours. The volatile materials were stripped off atreduced pressure. Water and benzene were added to the residue causingthe product to crystallize. The product was filtered, washed with waterand benzene and twice recrystallized from toluene with a carbontreatment each time; yield 8.9 g., m.p. l49. The infrared and nuclearmagnetic resonance spectra were fully consistent for the desiredproduct.

Anal. Calcd. for

Found: C, 4943;1-1, 3.54; N,

Sodium 6-[3-(p-chlorobenzyl)-1,2,4-oxadiazole-S-one-4-acetamidol-penicillanate A suspension of 4.0 g. of3-(p-chlorobenzyl)-1,2,4-oxadiazole-5-one-4-acetic acid in 200 ml. ofmethylene chloride was gassed for about 1 minute with hydrogen chloride.Phosphorus pentachloride (4.04 g.) was added (effervescence) and thereaction mixture was stored at room temperature for 2 hours withoccasional swirling. The solvent was stripped ofi at reduced pressure.The residual oil was triturated in the cold with Skellysolve B and driedin vacuo to remove the last traces of solvent leaving3-(p-chlorobenzyl)- 1,2,4-oxadiazole-5-one-4-acetyl chloride as residue.

A solution of the acid chloride in 90 ml. of acetone was added in oneportion to a rapidly stirred solution of of 3.23 g. of6-arninopenicillanic acid, 5.02 g. of sodium bicarbonate, ml. of waterand 60 ml. of acetone at room temperature. The reaction mixture wasstirred for one-half hour at room temperature and then extracted 3 timeswith ethyl acetate. The aqueous phase was layered with ethyl acetate andacidified with 42% phosphoric acid. Two more extractions with ethylacetate were made on the aqueous phase. The combined ethyl acetateextracts were washed 3 times with water, dried with sodium sulfate,filtered and treated with 5.7 ml. (0.01495 mole) of sodium2-ethylhexanoate in l-butanol giving a clear solution. The solvent wasstripped off at reduced pressure. The residue was triturated withanhydrous ether and the solid collected by filtration. The solid wasextracted with 150 ml. of acetone. The filtered acetone solution wasstripped of solvent. The residue was triturated with anhydrous ethergiving a solid; yield 4.7 g. after drying in vacuo over phosphoruspentoxide, decomposes l75180. The infrared and nuclear magneticresonance spectra were consistent for sodium6-[3-(p-chlorobenzyl)-1,2,4-oxadiazole-5-one-4- acetamido]penicillanate.

Found:

Found values corrected for 2.1% H 0:

6-[N-(p-Chlorophenylacetimidoyl)aminoacetamido1penicillanic acid Asolution of 4.5 g. of sodium 6-[3-(p-chlorobenzyl)- 1,2,4-oxadiazole--one-4-acetamidolpenicillanate in 200 ml. of water washydrogenated in the presence of commercial Raney nickel catalyst (No.28) on a Parr hydrogenation apparatus at an initial pressure of 50p.s.i. at room temperature for 1 hour. The catalyst was removed byfiltration. The filtrate was adjusted to pH 2.5 with 6 N hydrochloricacid and then to pH 4.2 with percent sodium hydroxide. The water wasstripped off at reduced pressure. The residue was further dried byadding ethyl acetate and then acetone and evaporating to dryness. Theresidue was extracted with 95 percent ethanol the insoluble materialbeing removed by filtration. To the filtrate was added about ml. ofacetone. The oily material which immediately separated was removed byfiltration. The clear filtrate was stored in the cold overnight causingthe product to crystallize. The product was filtered, washed with 1:1 95percent ethanol-acetone and with acetone and dried in a vacuum oven at60; yield 1.42 g., decomposes l65-l80. The infrared and nuclear magneticresonance spectra were consistent for the desired product.

EXAMPLE 4 3-Benzyl-l ,2,4-oxadiazole-5-one Phenylacetonitrile (235 g., 2moles) was added to a stirred mixture of 167 g. (2.4 moles) ofhydroxylamine hydrochloride 127 g. (1.2 mole) of sodium carbonate, 1 1of methanol and 600 ml. of water. The stirred mixture was heated at 50(solution) for 21 hours. The methanol was distilled off at reducedpressure. The crude amide oxime which separated as an oil was extractedinto 700 ml. of chloroform. The chloroform extract was washed 3 timeswith water, dried with sodium sulfate, filtered and about 200 ml. ofsolvent distilled ofi at reduced pressure to remove any remaining waterleaving a dry solution of phenylacetamide oxirne in chloroform.

Ethyl chlorofonnate (190 ml., 2 moles) was added in a slow stream to astirred and cooled (2530) solution of the above obtained chloroformsolution of phenylacetamide oxime, an additional 1.5 l of chloroform and280 ml. (2 moles) of triethyl amine. The solution was concentrated atreduced pressure (in 3 batches) until a thick residue of product andtriethylamine hydrochloride remained. The batches were combined andextracted 3 times with water. The chlorofonnproduct phase was strippedat reduced pressure to remove the remainder of the solvent. To theresidue was added a solution of 120 g. of sodium hydroxide in 1 1 ofwater. The vigorously stirred mixture was heated rapidly during about 10minutes to 70-8O causing most of the material to go into solution. Thecooled (25) mixture was extracted once with 300 ml. of benzene to removea small amount of insoluble oil. The ice cooled aqueous phase wasacidified (pH 2-3) with concentrated hydrochloric acid. The product wascollected by filtration, washed with water and air dried; yield 153.1 g.The product was suspended in 800 ml. of water and aqueous sodiumhydroxide solution (69 g. of sodium hydroxide in 200 ml. of water) addedin slight excess of the amount needed to dissolve all of the solid. Thesolution was carbon treated, filtered and acidified with concentratedhydrochloric acid. The product was filtered, washed with water and airdried; yield 119.6 g., m.p. ll1-1l4. 3-Benzyl- 1,2,4-oxadiazole-5-one-4-acetic acid A solution of 10 g. (0.057 mole) of3-benzyl-1,2,4-oxadiazole-S-one, 10.4 g. (0.0625 mole) of ethylbromoacetate and 8.8 ml. (0.0625 mole) of triethylamine in 100 ml. oftetrahydrofuran was stirred at room temperature for 5 hours. During thistime a thick precipitate of triethylamine hydrobromide formed. Afterstorage at room temperature for 64 hours the solid was removed byfiltration. The filtrate was concentrated at reduced pressure to removesolvent. The residue of crude ethyl3-benzyl-l,2,4-oxacliazole-5-one-4-acetate was combined with 80 ml. ofglacial acetic acid and ml. of 6 N hydrochloric acid and the solutionrefluxed for 1.5 hours. The

volatile materials were distilled off at reduced pressure. An infraredspectrum of the residue indicated that the hydrolysis of the ester wasincomplete. Therefore, ml. of glacial acetic acid and 40 ml. of 6 Nhydrochloric acid were added and the solution was refluxed for 2.5hours. The volatile materials were removed at reduced pressure. Waterand benzene were added to the residue and the mixture stored at roomtemperature overnight to crystallize the product. After cooling in anice bath the product was filtered and air dried. Recrystallization fromtoluene with a carbon treatment gave 8.0 g. (60 percent yield) of3-benzyl-l,2,4-oxadiazole-5-one-4-acetic acid; m.p. ll3115.

EXAMPLE 5 The procedures of Example 2 are repeated while replacing thebenzonitrile used therein with an equimolar weight of the nitrileprepared by P 0 dehydration of each of the corresponding amides havingthe following structures:

CONH:

\ and to produce, respectively, the compounds of the formulae Whennecessary the amide is prepared by converting the corresponding acid toits acid chloride with thionyl chloride and thence to the amide bytreatment of the acid chloride with ammonia in the usual manner.

EXAMPLE 6 The procedures of Example 2 are repeated while replacing thebenzonitrile used therein with an equimolar weight of the nitrile of theformula R CN wherein R is dichloromethyl, trichloromethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, cyclobutyl,cyclopentyl, cyclohexyl and cycloheptyl, respectively to produce,respectively, the cor-- responding compounds of the formulas wherein Zis (lower)alkyl, cycloalkyl having 4, 5, 6 or 7 carbon atoms, monohalo(lower)alkyl, dichloromethyl, trichloromethyl, (lower-)alkenyl of 2 to 6carbon atoms,

wherein R is hydrogen, hydroxy, amino, methyl, methoxy or chloro.

5. The compound having the formula 6. The compound having the formula 7.The compound having the formula 8. A nontoxic, pharmaceuticallyacceptable salt of the compound of claim 5.

9. The sodium salt of the compound of claim 5. 10. The potassium salt ofthe compound of claim 5. 11. The hydrochloride of the compound of claim5. 12. The zwitterion form of the compound of claim 5.

I! i i

2. A compound of claim 1 wherein Z is phenyl, benzyl, thenyl, thienyl,furyl or (lower)alkyl.
 3. A compound of claim 1 wherein Z is benzyl,2-thenyl or 3-thenyl.
 4. A compound of claim 1 wherein Z has thestructure wherein R is hydrogen, hydroxy, amino, methyl, methoxy orchloro.
 5. The compound having the formula
 6. The compound having theformula
 7. The compound having the formula
 8. A nontoxic,pharmaceutically acceptable salt of the compound of claim
 5. 9. Thesodium salt of the compound of claim
 5. 10. The potassium salt of thecompound of claim
 5. 11. The hydrochloride of the compound of claim 5.12. The zwitterion form of the compound of claim 5.